The long-objective of this research is to seek a better understanding of the basic mechanisms of action of nitrite inhalants, particularly in relation to their time-dependent effects. Nitrite inhalants ("poppers"), such as isobutyl nitrite (ISBN) and isoamyl nitrite (ISAN), are used mainly by male homosexuals, and also by teenagers, for their reported euphoric and aphrodisiac effects. A significant fraction of HIV-infected patients continues to use these substances of abuse. Nitrite abuse has been associated epidemiologically with the development of Kaposi's Sarcoma in AIDS patients, and tumor growth in animals. The basic pharmacology of these agents are poorly understood. In preliminary studies, we showed that ISBN inhalation/exposure caused several hitherto undiscovered effects: (1) up-regulation of genes encoding angiogenesis, e.g., that of vascular endothelial growth factor (VEGF), (2) induction of protein tyrosine nitration in liver and kidney tissues, (3) elevation of endothelial nitric oxide synthase (eNOS) protein in these tissues (4) significant reduction in the activities of several detoxifying hepatic enzymes. The induction of VEGF expression appears to be diminished in eNOS knockout mice. These studies revealed two novel mechanisms of nitrite toxicity, through gene regulation enhancing angiogenesis, and interrupting with the body's defense mechanisms against xenobiotics which may cause deleterious effects upon ingestion. Both of these effects may be time- and exposure-dependent, but these relationships are present unknown. In this proposal, we therefore seek to test the following hypotheses: (1) that the toxicokinetics and vasodilatory effects of inhalant nitrites are affected by repeated exposure, (2) that inhalant nitrites alter the regulation of angiogenesis genes, and this effect is governed by several factors, e.g., presence of eNOS, route of administration, and the tissues' oxidative state, (3) that repeated nitrite inhalations cause significant changes in the expression and activities of major hepatic detoxifying enzymes such as cytochrome P450s, glutathione-S-transferases. We shall use both rats and mice to examine these phenomena, with special attention toward the possible occurrence of pharmacological tolerance and withdrawal effects. We believe that our proposal represents novel approaches toward the understanding of nitrite inhalant toxicity in vivo.